Midline signalling systems direct the formation of a neural map by dendritic targeting in the Drosophila motor system.

Department of Zoology, University of Cambridge, Cambridge, United Kingdom.

Abstract

A fundamental strategy for organising connections in the nervous system is the formation of neural maps. Map formation has been most intensively studied in sensory systems where the central arrangement of axon terminals reflects the distribution of sensory neuron cell bodies in the periphery or the sensory modality. This straightforward link between anatomy and function has facilitated tremendous progress in identifying cellular and molecular mechanisms that underpin map development. Much less is known about the way in which networks that underlie locomotion are organised. We recently showed that in the Drosophila embryo, dendrites of motorneurons form a neural map, being arranged topographically in the antero-posterior axis to represent the distribution of their target muscles in the periphery. However, the way in which a dendritic myotopic map forms has not been resolved and whether postsynaptic dendrites are involved in establishing sets of connections has been relatively little explored. In this study, we show that motorneurons also form a myotopic map in a second neuropile axis, with respect to the ventral midline, and they achieve this by targeting their dendrites to distinct medio-lateral territories. We demonstrate that this map is "hard-wired"; that is, it forms in the absence of excitatory synaptic inputs or when presynaptic terminals have been displaced. We show that the midline signalling systems Slit/Robo and Netrin/Frazzled are the main molecular mechanisms that underlie dendritic targeting with respect to the midline. Robo and Frazzled are required cell-autonomously in motorneurons and the balance of their opposite actions determines the dendritic target territory. A quantitative analysis shows that dendritic morphology emerges as guidance cue receptors determine the distribution of the available dendrites, whose total length and branching frequency are specified by other cell intrinsic programmes. Our results suggest that the formation of dendritic myotopic maps in response to midline guidance cues may be a conserved strategy for organising connections in motor systems. We further propose that sets of connections may be specified, at least to a degree, by global patterning systems that deliver pre- and postsynaptic partner terminals to common "meeting regions."

Development of medio-lateral dendritic patterns between 15 h and 21 h AEL.

Single DiI/DiD-labelled dendritic trees of six representative motorneurons are shown at three different time-points during embryonic development (15 h, 18.5 h, and 21 h AEL). (B–E, H–K, N–Q) In most cases, dendritic territories characteristic for developmental stages when the motor system is functional (18.5 h AEL) have already become apparent by 15 h AEL. Curved arrows indicate dendrites in the intermediate territory and straight arrows dendrites in the medial neuropile. (A, G, M) MN-VO4/5 has “late exploring” dendrites that innervate the midline neuropile (straight arrow) between 15 h and 18.5 h AEL (“late exploration”; compare [A and G]; asterisk indicates absence of midline branches at 15 h AEL). (F, L, R) MN-DO2 dendrites on the other hand appear to undergo “late refinement” between 15 h and 18.5 h AEL. Branches are seen reaching into the intermediate/medial neuropile at 15 h AEL ([F, F′] straight arrow) but by 18.5 h AEL all MN-DO2 dendrites are confined to the lateral neuropile (“late refinement”; asterisks in [L and R] indicate intermediate/medial neuropile devoid of MN-DO2 dendrites at 18.5 h and 21 h AEL). Because the MN-DO2 cell body can obscure dendritic trees in projection views a cross section view is also shown in (F′) and optical slices that would have shown the ventrally located cell body have been omitted from the projection shown in (R) so that the dendrites can be seen clearly. In general, motorneuron dendritic trees have markedly increased in size from 15 h to 18.5 h AEL. Between 18.5 h and 21 h AEL (hatching) further adjustments in dendritic extent, morphology, and position occur but are more subtle. For instance, at 18.5 h AEL 7/9 labelled MN-VO4/5 show posteriorly projecting dendritic branches to varying degrees (see [G] for an example with a pronounced posterior dendritic projection) that are not seen at 21 h AEL (n = 6). In all micrographs anterior is up except for (F′) where dorsal is up. Dotted lines indicate CNS midlines. Scale bar: 20 µm.

Medio-lateral dendritic targeting in the absence of cholinergic synaptic transmission.

Single DiI/DiD-labelled dendritic trees of four representative motorneurons (as described in ) are shown at 18.5 h AEL in wild-type and cha mutant embryos. (A, E) By 18.5 h AEL, both in the control and mutant condition, MN-VO4/5 has established its characteristic medial dendritic subtree (straight arrow), respectively (n = 9). (D, H) MN-DO2 (n = 3) and MN-DO1 (n = 10; not shown) dendrites are strictly confined to the lateral neuropile in cha mutant embryos as in the wild-type (note that in [D] the “common exciter” RP2 was also labelled). (B, C) MN-LL1 and MN-DA3 can normally be clearly distinguished: MN-LL1 has manifest branches innervating the intermediate neuropile (curved arrow), which are not formed by MN-DA3 (asterisk). (F, G) In 18.5 h cha mutants, some MN-LL1 and MN-DA3 cells form dendrites that are less distinct than in the wild-type and examples of such cases are shown here: in 3/10 cases MN-LL1 dendrites in the intermediate territory were less extensive than in the wild-type (curved arrow in [F], compare with [B and G]); in 4/11 cases MN-DA3 dendrites extended slightly more medially than in controls (curved arrow in [G], compare with [C and F]). Asterisks in (C, D, and H) indicate intermediate neuropile devoid of dendritic branches. Dotted lines indicate CNS midlines. Scale bar: 20 µm.

Normal positioning and contact with presynaptic cholinergic terminals is not required for dendritic medio-lateral targeting.

(A) Digital cross section from a confocal stack and a schematic cross section (A′) of a motorneuron labelled with DiD (yellow) in the ventral nerve cord relative to the Fasciclin2-GFP-positive tracts (cyan) at 18.5 h AEL. The motorneuron dendrites arborise in the dorsal (motor) neuropile whereas sensory axons primarily terminate in the ventral neuropile. (B–D′) show corresponding cross sections of nerve cords in which cholinergic processes are visualised by expression of membrane targeted UAS-myr-mRFP using Cha-GAL4 (red) in the context of Fasciclin2-GFP-positive tracts (cyan). In addition, chimeric Robo-Fra receptors were expressed to displace the cholinergic terminals (C–D′). (C, C′) Expression of the RoboexFrain receptor leads to a thickening of the commissural cholinergic tracts ventrally (arrowheads) and an accumulation of cholinergic terminals dorsally at the midline (straight arrows). (D, D′) Conversely, expression of the repulsive receptor FraexRoboin induces a severe depletion of cholinergic fibers from the commissures (asterisks). (E–G) Single confocal slices at a position in the dorsal neuropile where motorneuron dendrites (labelled with DiD, green) form show a marked decrease in cholinergic innervation (red) when the chimeric receptors are expressed (compare [E] with [F and G]; note that in [G] the dendritic tree was extremely brightly labelled resulting in a fraction of the DiD signal being picked up in the red RFP channel so that the overlay of both channels appears yellow in places). (H–J) Cumulative plots of MN-LL1 and MN-DA3 dendritic trees in controls (H) and under experimental conditions (I, J) show that their medio-lateral distinctions are clearly apparent, as in the wild-type ([H], n-numbers given in parentheses). Unlike MN-DA3, MN-LL1 reproducibly targets the intermediate neuropile (white curved arrows in [H–J]). However, in 4/7 cases MN-LL1 formed abnormal posteriorly projecting branches in the intermediate neuropile in Cha-GAL4; UAS-FraexRoboin embryos (black curved arrow in [J]). D, dorsal; V, ventral; A, anterior; P, posterior. Dotted lines indicate CNS midlines. Scale bar: 20 µm.

Frazzled and Robo are required cell-autonomously and gate dendritic targeting to the midline.

Single DiI/DiD-labelled dendritic trees of MN-DA3, MN-LL1, and MN-VO4–6 at 18.5 h AEL in controls and when levels of Frazzled and/or Robo have been altered. The midline targeting dendrite of MN-VO4–6 ([A] straight arrow) fails to form when UAS-robo is expressed (using CQ-GAL4 [D]) or in a fra-mutant background (G). The fra-mutant phenotype can be rescued by reinstating UAS-fra with CQ-GAL4 ([J] straight arrow). Conversely, in MN-DA3 and MN-LL1 (B and C for wild-type) inactivation of Robo (CQ-GAL4 driving UAS-comm or robo1/roboGA1112) produces a usually single aberrant midline targeting dendritic branch per cell ([E, F, H, I] straight arrows). The robo-mutant phenotype can be rescued by driving UAS-robo with CQ-GAL4 (K, L). The UAS-comm phenotype is suppressed in a fra-mutant background (asterisks in [M, N] indicate the absence of midline targeting branches; compare with [E, F]) and recovered by co-expressing additionally UAS-fra in MN-DA3 and MN-LL1 ([O, P] straight arrows). (Q) illustrates the distribution of dendritic phenotypes for each motorneuron and genotype with indicated n-numbers above each bar. Dotted line: CNS midline. Scale bar: 20 µm.

Robo and Frazzled mediate dendritic targeting in the intermediate and lateral neuropile.

(A–F) DiI/DiD-labellings of single MN-LL1 and MN-DA3 at 18.5 h AEL in fra- or robo-manipulated genetic backgrounds. (A′–F′) Cumulative plots generated from z-projections of various cells that were mapped onto a common reference grid using Fasciclin2-GFP-positive axon bundles as landmarks and shown in two channels, each representing one of two experimental conditions (n-numbers of cells in each plot are given in parentheses). Saturated colours indicate highly reproducible dendritic coverage at the respective relative position. In the wild-type, MN-LL1 can be distinguished from MN-DA3 by the presence of a dendritic subtree located in the intermediate neuropile (white curved arrow in [A, A′]). (B–C′) In fra mutants (C, C′) and when UAS-robo is expressed (B, B′; using CQ-GAL4) the intermediate dendrites of MN-LL1 fail to form (asterisks in [B and C]). (D, D′) Cell-specific expression of UAS-fra in the fra-mutant background rescues the intermediate MN-LL1 dendrites (white curved arrows) and frequently generates an ectopic posteriorly projecting intermediate branch (black curved arrows). (E, E′) Similarly, expression of UAS-fra in a wild-type background produces ectopic posteriorly projecting intermediate dendrites in MN-LL1 (black curved arrows). (F, F′) UAS-fra expression in MN-DA3 results in a subtle ectopic innervation of the intermediate neuropile (white curved arrows). (G) Illustration of and penetrance of four distinct medio-lateral dendritic morphologies for each motorneuron and genotype with indicated n-numbers above each bar. Dotted line: CNS midline. Scale bar 20 µm.

Dendritic targeting phenotypes are not associated with overall changes in dendritic length and tip numbers.

Images of representative digitally reconstructed dendritic trees (beige; axons coloured red) of MN-LL1 (A–D) and MN-DA3 (E, F) in different genetic backgrounds (18.5 h AEL; transgenes were expressed using CQ-GAL4; n-numbers are given in parentheses). (G) Box plots illustrating the distribution of total dendritic tree length and dendritic tip number. The median is indicated by a thick black horizontal bar, and the 25th and 75th percentiles are the bottom and top line of each box, respectively. Whiskers show the extremes of each dataset and in two cases outliers are indicated by circles. Although the medio-lateral positions of the dendritic trees vary dramatically depending on the genotype, their overall lengths and tip numbers do not show significant differences (G). Student's t test and Wilcoxon test were used for statistical analysis as appropriate. Anterior is up. Dotted line: CNS midline. Scale bar 20 µm.

Genetic manipulations of Robo in MN-LL1 lead to a redistribution of dendritic branches.

Images of representative digitally reconstructed dendritic trees of MN-LL1 under different experimental conditions ([A–C] 18.5 h AEL; transgenes were expressed using CQ-GAL4; n-numbers are given in parentheses). Each dendritic tree was subdivided into a lateral (yellow) and an intermediate/medial part (cyan) bisected by the intermediate Fasciclin2-GFP-positive tracts ([A–C] arrowheads). (D) Lengths of laterally located dendrites and total tree lengths were quantified individually and the ratios of lateral/total tree length are shown using box plots. The median is indicated by a thick black horizontal bar, and the 25th and 75th percentiles are the bottom and top line of each box, respectively. Whiskers show the extremes of each dataset and in two cases outliers are indicated by circles. Down-regulation of Robo by expression of UAS-comm in MN-LL1 induces ectopic dendritic innervation of the medial neuropile ([A] cyan) and concomitantly leads to a decrease in the extent of the arbor located laterally ([A] yellow). Conversely, expression of UAS-robo elicits the opposite effect, a reduction of the arbor that innervates the intermediate territory ([C] cyan) and an increase in the extent of the tree in the lateral neuropile ([C] yellow). Student's t test was used for statistical analysis. Anterior is up. Dotted line: CNS midline. Scale bar 20 µm.

(A) Single confocal section of a motorneuron in freshly hatched larvae (21 h AEL) retrogradely labelled with DiD (cyan) with cholinergic presynaptic sites visualised with Cha-GAL4; UAS-brp-RFP (red). Part of the arbor (stippled outline) is shown enlarged in the inset in the bottom left-hand corner. (A′) The same confocal section is shown as in (A) and superimposed is a digital 3-D reconstruction of the entire dendritic arbor. Relative probabilities of synaptic connections were mapped onto the reconstructed arbour; colours towards the red spectrum indicating high probabilities based on brp-RFP fluorescence signal intensity and distance to dendrites (<400 nm). The insets in (A and A′) show an enlarged view of brp-RFP puncta and DiD-labelled dendrite in close apposition. (B) Dorsal views (a ventral view of part of the MN-LL1 arbor is shown in the inset) of representative reconstructed dendritic trees from dye-labelled MN-DA3, MN-LL1, and MN-VO4/5 with the distribution of putative synaptic sites mapped onto these as illustrated in (A and A′). Putative synaptic contacts (arrowheads) can be found on medial, intermediate, and lateral dendritic branches. Anterior is up. Scale bar: 5 µm in (A, A′) (2.5 µm for insets); 10 µm in (B).